U.S. patent number 7,279,497 [Application Number 10/523,015] was granted by the patent office on 2007-10-09 for benzopyran derivatives substituted with secondary amines including imidazole, their preparation and pharmaceutical compositions containing them.
This patent grant is currently assigned to Korea Research Institute of Chemical Technology. Invention is credited to Sang-Un Choi, Sun Kyung Hwang, Nak Jeong Kim, Byung-Ho Lee, Chong Ock Lee, Sun Kyung Lee, Ho Won Seo, Jee Hee Suh, Kyu Yang Yi, Sung-Eun Yoo.
United States Patent |
7,279,497 |
Yi , et al. |
October 9, 2007 |
Benzopyran derivatives substituted with secondary amines including
imidazole, their preparation and pharmaceutical compositions
containing them
Abstract
The present invention relates to benzopyran derivatives
substituted with secondary amines including imidazole, their
preparation, and pharmaceutical compositions containing them. The
present invention is pharmacologically useful for the treatment of
cancer, rheumatoid arthritis, and diabetic retinopathies through
anti-angiogenic properties, and also pharmacologically useful in
the protection of heart and neuronal cells against
ischemia-reperfusion injury or preserving organs.
Inventors: |
Yi; Kyu Yang (Taejeon-si,
KR), Lee; Sun Kyung (Taejeon-si, KR), Yoo;
Sung-Eun (Gongju-si, KR), Suh; Jee Hee
(Taejeon-si, KR), Kim; Nak Jeong (Taejeon-si,
KR), Hwang; Sun Kyung (Taejeon-si, KR),
Lee; Byung-Ho (Taejeon-si, KR), Seo; Ho Won
(Taejeon-si, KR), Lee; Chong Ock (Taejeon-si,
KR), Choi; Sang-Un (Taejeon-si, KR) |
Assignee: |
Korea Research Institute of
Chemical Technology (Taejeon-si, KR)
|
Family
ID: |
36165420 |
Appl.
No.: |
10/523,015 |
Filed: |
July 30, 2003 |
PCT
Filed: |
July 30, 2003 |
PCT No.: |
PCT/KR03/01534 |
371(c)(1),(2),(4) Date: |
February 02, 2005 |
PCT
Pub. No.: |
WO2004/014898 |
PCT
Pub. Date: |
February 19, 2004 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20050267188 A1 |
Dec 1, 2005 |
|
Foreign Application Priority Data
|
|
|
|
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Aug 9, 2002 [KR] |
|
|
10-2002-0047189 |
|
Current U.S.
Class: |
514/397;
548/311.1; 548/300.1; 514/396; 548/311.4; 514/385 |
Current CPC
Class: |
C07D
405/12 (20130101); A61P 9/04 (20180101); A61P
25/02 (20180101); A61P 39/06 (20180101); A61P
43/00 (20180101); A61P 9/00 (20180101); A61P
25/28 (20180101); A61P 19/02 (20180101); A61P
25/00 (20180101); A61P 29/00 (20180101); A61P
35/00 (20180101); A61P 9/10 (20180101); A61P
27/06 (20180101); A61P 27/02 (20180101) |
Current International
Class: |
A61K
31/4164 (20060101); C07D 233/54 (20060101) |
Field of
Search: |
;548/300.1,311.1,311.4
;514/385,396,397 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
5629429 |
May 1997 |
Kronenthal et al. |
5837702 |
November 1998 |
Rovnyak et al. |
5869478 |
February 1999 |
Ding et al. |
6521617 |
February 2003 |
Marban et al. |
|
Foreign Patent Documents
Other References
Grover, G.J., et al., "Pharmacologic Characterization of
BMS-191095, a Mitochondrial K.sub.ATP Opener with No Peripheral
Vasodilator or Cardiac Action Potential Shortening Activity", The
Journal of Pharmacology and Experimental Therapeutics, vol. 297,
No. 3, pp. 1184-1192, (2001). cited by other .
Ding, C.Z., et al., "Cardio selective Antiischemic ATP-Sensitive
Potassium Channel (K.sub.ATP) Openers. 6. Effect of Modifications
at C6 of Benzopyranyl Cyanoguanidines", Journal of Medicinal
Chemistry, vol. 42, No. 18, pp. 3711-3717, (1999). cited by other
.
Rovnyak, G.C., et al., "Cardio selective Antiischemic ATP-Sensitive
Potassium Channel (K.sub.ATP) Openers. 5. Identification of
4-(N-Aryl)-Substituted Benzopyran Derivatives with High
Selectivity", Journal of Medicinal Chemistry, vol. 40, No. 1, pp.
24-34, (1997). cited by other.
|
Primary Examiner: Shameem; Golam M. M.
Attorney, Agent or Firm: Nath; Gary M. Goldberg; Joshua B.
Harkins; Tanya E.
Claims
What is claimed is:
1. Benzopyran derivatives substituted with secondary amines
including imidazole by the following formula 1, their
stereochemical isomers and their pharmaceutically acceptable salts:
##STR00010## Wherein, R.sup.1 represents H, CN, NO.sub.2 or
NH.sub.2, R.sup.2 represents ##STR00011## wherein R.sup.a
represents straight or branched alkyl group of C.sub.1-C.sub.4,
R.sub.3 and R.sub.4 are independent each other and represent H, Cl,
Br, F, alkyl group of C.sub.1-C.sub.3, OR.sup.b, CF.sub.3,
OCF.sub.3, NO.sub.2, or CO.sub.2R.sup.b; R.sup.b represents H or
alkyl group of C.sub.1-C.sub.3, and * represents the chiral
center.
2. Benzopyran derivatives substituted with secondary amines
including imidazole, their stereochemical isomers and their
pharmaceutically acceptable salts according to claim 1, wherein the
compound of formula 1 is selected from the group consisting of: 1)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
2)
(2S,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
3)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
4)
(2R,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
5)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-trifluoromethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran-
; 6)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
7)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyra-
n; 8)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro--
4-[N-(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
9)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2,4-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
10)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2-isopropylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
11)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
12)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
13)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
14)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
15)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-fluorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
16)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
17)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2-isopropylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
18)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
19)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(3-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
20)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(3-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
21)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyra-
n; 22)
(2S,3S,4R)-6-cyano-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-
-4-[N-(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
23)
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
24)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
25)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-trifluoromethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran-
; 26)
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl--
4-[N-(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benz-
opyran; 27)
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
28)
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
29)
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
30)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
31)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(2-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
32)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
33)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(2,4-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
34)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(2-isopropylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
35)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyra-
n; 36)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-
-4-[N-(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
and 37)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-meth-
yl-4-[N-(4-fluorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran.
3. A process for preparing the benzopyran derivatives substituted
with secondary amines including imidazole of claim 1, comprising
the step of reacting an epoxide compound (II) with a secondary
amine compound including imidazole (III) in the presence of a metal
salt in an reaction solvent to obtain a compound (I), as described
in scheme 1: ##STR00012## Wherein R.sub.1, R.sub.2, R.sub.3,
R.sub.4 * and n are each defined as above claim 1, the metal salt
is selected from the group consisting of Mg(ClO.sub.4).sub.2,
CoCl.sub.2, LiClO4.sub.2, NaClO.sub.4, CaCl.sub.2, ZnCl.sub.2,
LiBF.sub.4 and Zn(Tf).sub.2, and the reaction solvent is selected
from the group consisting of acetonitrile, tetrahydrofuran and
dimethylformamide.
4. A process for for preparing the benzopyran derivatives
substituted with secondary amines including imidazole of claim 1,
comprising the step of 1) reduction of the nitro compounds (IV) by
hydrogenation using metal catalysts such as platinum, palladium,
palladium on carbon (Pd/C), Raney-nickel, etc. in a suitable
solvent, to obtain the amino compound (V) as described in scheme 4,
below; or 2) reduction of the nitro compounds (IV) using an
reducing agent in the presence of CuSO.sub.4, Cu(OAc).sub.2,
CoCl.sub.2, SnCl.sub.2 or NiCl.sub.2, to obtain the amino compound
(V) as described in scheme 4, below: ##STR00013## Wherein R.sup.2,
R.sup.3, R.sup.4 and * are each defined as above claim 1.
5. The process according to claim 4, wherein the reducing agent is
NaBH.sub.4.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel benzopyran derivatives
substituted with secondary amines including imidazole of formula 1.
It also relates to process for preparing the novel compounds and
pharmaceutical formulations comprising one or more of the compounds
as an active ingredient.
The present invention also relates to pharmaceutical use of the
benzopyran derivatives substituted with secondary amines including
imidazole. In particular, the present invention is
pharmacologically useful in the treatment of cancer, rheumatoid
arthritis, and diabetic retinopathies through anti-angiogenic
properties, and also pharmacologically useful for the protection of
heart, neuronal cells, brain injury, organs for preservation or in
major cardiovascular surgery against ischemia-reperfusion injury or
oxidative stress.
##STR00001##
Wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and * are each defined
in specification.
2. Description of the Prior Art
The ratio of cancer in human diseases is being gradually increased
despite the considerable research has been devoted to the whole
area of cancer. Since the 1970s discovery by J. Folkman that
angiogenesis, the formation of new blood vessels from preexisting
vessels, is implicated in tumor growth, anti-angiogenics have been
identified as one of the most promising and innovative drug
classes.
Traditional chemotherapeutics destroy tumor cell populations by
chemical poisoning of cancer cells during their productive cycles,
which affect normal cells as well as tumor cells resulting in
serious side effects. Therefore, the research on the development of
anti-angiogenic agents, which inhibit the formation of new blood
vessels to provide oxygen and nutrients, and to provide a way to
metastasize to distant organs, is considered as one of the novel
approaches for the anti-cancer therapies.
While angiogenesis normally occurs in adults only in the specific
conditions such as wound healing and inflammation, angiogenesis is
recognized as the core process for growth and metastasis of solid
tumors because solid tumors could only grow to 1-2 mm without
developing a blood supply (Folkman, J. et al., J. Biol. Chem. 267:
10931-10934 (1992)). In normal conditions the angiogenic process is
under tight regulation of stimulatory and inhibitory factors. Under
certain pathological conditions such as the growth of solid tumors,
rheumatoid arthritis, psoriasis, complications of AIDS, and
diabetic retinopathy, angiogenesis occurs in a less controlled
manner (Forkman, J., Klagsbrun. M. Science 235: 442-447 (1987).
Angiogenesis includes a series of processes such as the migration,
proliferation and differentiation of endothelial cells, and is an
important prerequisite for the growth and metastasis of cancers. In
details because the growing tumor cells require the formation of
blood vessels from host cells, angiogenesis promoters derived from
tumors stimulate to induce the angiogenesis into the tumor mass.
Afterwards, the blood vessels formed around the malignant tumors
facilitate to metastasize the tumor cells to other sites.
Therefore, the inhibition of angiogenesis leads to the prevention
of the growth and metastasis of cancers. As one of the important
research areas for the developing of anti-cancer drugs, extensive
attention is paid to the finding of angiogenesis inducers and
angiogenesis inhibitors and the revealing of their working
mechanisms.
Because angiogenesis is a complex process with multiple, sequential
and independent steps, it creates many potential targets for
inhibition, including inhibition of angiogenesis inducers'
production, inhibition of the binding of angiogenesis inducers to
their receptors, inhibition of basal membrane degradation,
inhibition of endothelial proliferation and migration, inhibition
of capillary tube formation, and inhibition of basal membranes'
syntheses and migration, etc. Thus far, proteins such as prostamine
and tumor necrotic factors, polysaccharides, antibiotics, various
steroid derivatives, polycataions, and polyanions have been found
to be able to play roles as angiogenesis inhibitors. In particular,
hydrocortisone exhibits anti-angiogenetic activity by cotreatment
with heparin (Lee, A. et al., Science 221: 1185-1187 (1983); Crum,
R. et al., Science 230: 1375-1378 (1985)). Recently Astra Zeneca's
Iressa was launched for non small cell lung carcinoma, and several
anti-angiogenic agents are currently in clinical trials. Neovastat,
Tarceva, CAI and Thalomid are under phase III clinical trials with
some positive results.
Ischemic heart diseases usually occur as a result of myocardial
ischemia, when the oxygen supply is significantly decreased
compared to the oxygen demand due to the imbalance between them. In
most cases, a coronary artery disorder was found to be a main
reason of the ischemic heart diseases. If the inner diameter of
coronary artery becomes narrow, the blood supply, resulting in
oxygen supply, becomes insufficient, which can cause angina
pectoris, myocardial infarction, acute cardioplegia, arrhythmia,
and so on (G. J. Grover, Can. J. Physiol. 75, 309 (1997); G. D.
Lopaschuk et al., Science & Medicine 42 (1997)). Because
ischemic heart diseases are also caused by other complex factors
besides coronary artery disorders, drug therapy as well as
operational method such as percutaneous transluminal coronary
angioplasty (PTCA) is required for its treatment. For that purpose,
several drugs are being used, including anti-thrombotic agents,
arteriosclerosis, curatives, especially beta blockers, nitrate,
calcium antagonists such as nifedipin, thromobolytics, aspirin, and
angiotensin converting enzyme (ACE) inhibitors.
Differently from conventional potassium channel openers, the
benzopyranyl anilinomethylimidazole compound (BMS-191095), has been
reported to act selectively on ATP-sensitive potassium channels
(K.sub.ATP) located in the heart (K. S. Atwal et al., J. Mde. Chem.
36, 3971 (1993); K. S. Atwal et al., J. Me. Chem. 38, 1966 (1995)).
The BMS 191095 compound was found to protect ischemic hearts
without a significant lowering of blood pressure, which gives the
prospects for novel drug development as a cardioprotectant.
##STR00002##
Damage or death of neurons is known to be a main cause for various
neurological disorders such as stroke, head trauma, Alzheimer's
disease, Parkinson's disease, infant asphyxia, glaucoma and
daiabetic neuropathy, etc. (G. J. Zoppo et al., Drugs 54, 9(1997);
I. Sziraki et al., Neurosci. 85, 110(1998)). Neurons are damaged by
various factors and typically by increases in iron concentration,
reactive oxygen species, and peroxidants within neurons (M. P.
Mattson et al., Methods Cell Biol. 46; 187 (1995); Y. Goodman et
al., Brain Res. 706, 328 (1996)).
The intensive research on the development of compounds with the
above-mentioned pharmacological efficacies by the inventors, found
that the benzopyran derivatives substituted with secondary amines
including imidazole represented by the formula 1. The compounds
exhibit various pharmacological efficacies, including suppression
of angiogenesis, in vivo anti-cancer activity, cardioprotection
against ischemia-reperfusion injury, neuroprotective activity,
prevention of lipid peroxidation and reactive oxygen species
formation. Thus the compound of the present invention can be useful
in the prevention and treatment of various diseases related to
angiogenesis such as cancers, rheumatoid arthritis, and diabetic
retinopathy; neuronal damage such as infant asphyxia, glaucoma,
diabetic neuropathy and head trauma; oxygen free radical-related
disease such as neurodegenerative diseases and atherosclerosis; and
diseases related to cardiovascular system such as myocardial
infarction, congestive heart failure, and angina pectoris.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides benzopyran derivatives substituted
with secondary amines including imidazole by the following formula
1 and their pharmaceutically acceptable salts.
##STR00003## Wherein R.sup.1 represents H, CN, NO.sub.2 or
NH.sub.2, R.sup.2 represents CH.sub.3,
##STR00004## R.sup.a represents straight or branched alkyl group of
C.sub.1-C.sub.4; and Z is straight or branched alkyl group of
C.sub.2-C.sub.6, R.sup.3 and R.sup.4 are independent each other and
represent H, Cl, Br, F, alkyl group of C.sub.1-C.sub.3, OR.sup.b,
CF.sub.3, QCF.sub.3, NO.sub.2, or CO.sub.2R.sup.b; R.sup.b
represents H or alkyl group of C.sub.1-C.sub.3, and * represents
the chiral center.
The present invention includes all the solvates and hydrates which
can be prepared from benzopyran derivatives substituted with
secondary amines including imidazole of formula 1 in addition to
benzopyran derivatives substituted with secondary amines including
imidazole of formula 1 and their pharmaceutically acceptable
salts.
The present invention includes all the separate stereochemical
isomers, i.e. diastereomerically pure or enantiomerically pure
compounds which have one or more chiral centers at 2, 3 and
4-positions, in addition to the racemic mixtures or diastereomer
mixtures of benzopyran derivatives substituted with secondary
amines including imidazole of formula 1.
In case of having three chiral centers at 2, 3 and 4-positions, the
3,4-dihydrobenzopyran derivatives according to the present
invention are represented by the optical isomers such as (I.sub.1),
(I.sub.2), (I.sub.3) and (I.sub.4). (See the following formula
2).
##STR00005##
Wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are defined as
above.
In particular, the preferable compounds of the present invention
are:
1)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4--
[N-(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
2)
(2S,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4--
[N-(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
3)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4--
[N-(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
4)
(2R,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4--
[N-(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
5)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4--
[N-(4-trifluoromethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopy-
ran;
6)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4--
[N-(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
7)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4--
[N-(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzop-
yran;
8)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4--
[N-(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
9)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4--
[N-(2,4-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
10)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(2-isopropylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
11)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
12)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
13)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
14)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
15)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(4-fluorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
16)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(2-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
17)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(2-isopropylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
18)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(2-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
19)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(3-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
20)
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(3-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
21)
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-
-[N-(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzo-
pyran;
22)
(2S,3S,4R)-6-cyano-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
23)
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
24)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
25)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-trifluoromethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzop-
yran;
26)
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzo-
pyran;
27)
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
28)
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
29)
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
30)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
31)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(2-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
32)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
33)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(2,4-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
34)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(2-isopropylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
35)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzo-
pyran;
36)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran;
and
37)
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-
-[N-(4-fluorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran.
The compounds of formula 1 may be used as pharmaceutically
acceptable salts derived from pharmaceutically or physiologically
acceptable free acids. These salts include but are not limited to
the following: salts with inorganic acids such as hydrochloric
acid, hydrobromic acid, sulfuric acid, sulfonic acid, phosphoric
acid, etc. and organic acids such as citric acid, acetic acid,
maleic acid, fumaric acid, gluconic acid, methanesulfonic acid,
glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic
acid, galacturonic acid, embonic acid, glutamic acid, aspartic
acid, etc.
The acid salts of the compounds according to the present invention
can be prepared in the customary manner, for example by dissolving
the compound of formula 1 in excess aqueous acid and precipitating
the salt with a water-miscible organic solvent, such as methanol,
ethanol, acetone or acetonitrile. It is also possible to prepare by
heating equivalent amounts of the compound of formula 1 and an acid
in water or an alcohol, such as glycol monomethyl ether, and then
evaporating the mixture to dryness or filtering off the
precipitated salt with suction.
In addition, the present invention provides processes for preparing
of the benzopyran derivatives substituted with secondary amines
including imidazole of formula 1.
In particular, the present invention provides processes for
preparing of the benzopyran derivatives substituted with secondary
amines including imidazole of formula 1 by the reaction of the
compound of formula II and the compound of formula III in the
presence of metal salt as represented in the following scheme
1.
##STR00006##
Wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, * and n are each
defined as above.
The derivatives of formula 1 can be prepared separately as an
optically active isomer by using the corresponding optical isomer
as a starting material.
In case of using a racemic mixture as a starting material, the
derivatives of formula 1 are prepared as a racemic mixture, and
then the racemic mixture is separated into each optical isomers.
The optical isomers can be separated by common chiral column
chromatography or recrystallization.
The compounds of formula 1 can be synthesized using the reactions
and techniques described herein below. The reactions are performed
in a solvent appropriate to the reagents and materials employed and
suitable for the transformation being effected.
I. Preparation of Starting Materials
(1) Preparation of epoxide compounds (II)
Epoxide compounds (II.sub.1) and epoxide compounds (II.sub.2) can
be prepared from the olefin compound (IV.sub.1) and epoxide
compounds (II.sub.3) and epoxide compounds (II.sub.4) can be
prepared from the olefin Compound (IV.sub.2) as represented by the
following scheme 2, by the method disclosed in KR Pat. Appln. No.
2000-60467 which was axquired by the present inventors.
##STR00007##
Wherein R.sup.1 and R.sup.2 are each defined as above formula
1.
(2) Preparation of Secondary Amine Compounds Including Imidazole
(III)
Secondary amine compounds including imidazole ring (III), which
were used in scheme 1, can be prepared by reductive amination of
2-imidazolecarboxaldehyde and aniline compounds as represented in
scheme 3.
##STR00008##
Wherein R.sup.3 and R.sup.4 are each defined as above formula
1.
In the above scheme 3, various reducing agents can be employed for
reductive amination such as sodium borohydride and sodium
cyanoborohydride, etc.
Preferred solvents are alcohols such as methanol, ethanol, etc., or
ethyl acetate.
Reaction temperature is preferably maintained from room temperature
to the boiling point of solvent employed.
II. Preparation Method
The method for the preparation of compounds (formula 1) comprises
the step of reaction of an epoxide compound (II) with secondary
amine compound (III) in the presence of proper metal salts.
Metal salts used for this reaction, are Mg(ClO.sub.4).sub.2,
CoCl.sub.2, LiClO.sub.4, NaClO.sub.4, CaCl.sub.2, znCl.sub.2,
LiBF.sub.4, Zn(Tf).sub.2, etc., and preferable solvents are
acetonitrile, tetrahydrofuran, dimethylformamide, etc. Reaction
temperature may range from room temperature to boiling point of
employed solvent.
In case of using each stereoisomer of the epoxide compound (II) as
a starting material, the product with the same configuration to
that of the starting material is obtained, respectively. That is,
the compounds (I.sub.1), (I.sub.2), (I.sub.3) or (I.sub.4) of
formula 1, can be prepared from epoxide compounds (II.sub.1),
(II.sub.2), (II.sub.3) or (II.sub.4) with amine compounds (III),
respectively.
The compounds (V) of formula 1 whose R.sub.1 is NH.sub.2 can be
prepared by the reduction of the compounds (IV) of which R.sup.1 is
NO.sub.2 as represented in the below scheme 4.
##STR00009##
Wherein R.sup.2, R.sup.3, R.sup.4 and * are each defined as
above.
The NO.sub.2 group can be reduced to NH.sub.2 group by
hydrogenation using metal catalysts such as platinum, palladium,
palladium on carbon (Pd/C), Raney-nickel, etc. in a suitable
solvent. Preferred solvents are alcohols such as methanol, ethanol,
etc., and ethyl acetate.
In addition, the reduction of NO.sub.2 group to NH.sub.2 group can
be carried out by using a reducing agent such as NaBH.sub.4, etc.
in the presence of CuSO.sub.4, Cu(OAc).sub.2, CoCl.sub.2,
SnCl.sub.2 or NiCl.sub.2, etc. At this time, preferred solvent is a
mixture of water and methanol and reaction temperature is from room
temperature to boiling point of employed solvent.
In addition, the present invention provides pharmaceutical
compositions which contain the benzopyran derivatives substituted
with secondary amines including imidazole of the above formula 1 or
their pharmaceutically acceptable salts as an active ingredient. In
particular, the present invention provides pharmaceutical
compositions for suppression of angiogenesis, protection of
neuronal cells, brain injury, heart, and organs for preservation or
during cardiovascular surgery, and antioxidants.
The compounds of the present invention have an ability to suppress
angiogenesis. In detail, the compounds of the present invention
inhibit the HUVEC (Human Umbilical Vein Endothelial Cell) tube
formation induced by bFGF (basic Fibroblast Growth Factor), and
suppress in vivo angiogenesis in mouse matrigel plug assay
(subcutaneous and oral administration) and CAM (chorioallantonic
membrane assay). Also, the compounds of present invention
significantly suppress the tumor growth in nude mouse xenografts of
A549 human non small cell lung carcinoma without any significant
side effects such as loss of body weights. No mice treated with the
compounds of present invention were died, which shows reduced
toxicity compared to traditional cytotoxic anti-cancer agents.
Therefore, the compounds of present invention can be used for
anti-cancer agents and can be applied for the treatment of
rheumatoid arthritis and diabetic retinopathies.
In addition, the compounds of the present invention have an ability
to protect neurons. In particular, the compounds of the present
invention protect neurons from oxidative stress induced by iron and
from neuronal cell damage induced by hydrogen peroxide. Therefore,
the compounds of the present invention can be used as a
neuroprotective and can also be applied for the prevention and
treatment of infant asphyxia, glaucoma, diabetic neuropathy, and
head trauma caused by neuronal cell damage or death.
Furthermore, the compounds of the present invention inhibit the
lipid peroxidation induced by iron or copper, and suppress
intracellular reactive oxygen species in A7r5 (Rat thoracic aorta
smooth muscle cell line, ATCC) induced by H.sub.2O.sub.2. Hence,
the compounds of the present invention can be used as an
antioxidant and can be effectively applied for the medical
treatment of the neurodegenerative disorders caused by lipid
peroxydation and the accumulation of free radical species within
neurons, such as aging and senile dementia.
In isolated ischemic rat heart model using Langendorff apparatus,
the compounds of the present invention significantly prolong the
time to contracture (TTC, time to contracture), improve the
recovery of postischemic contractile function (LVDP.times.HR, (left
ventricular developing pressure).times.(heart rat)), and decrease
the release of lactate dehydrogenase (LDH) which is a marker enzyme
for cell injury, then show similar cardioprotection effect compared
to that of BMS-180448. In addition, the compounds of the present
invention have low vasorelaxant activity in contrast to BMS-180448
and BMS-191095. Further, the compounds of the present invention
exhibited equal antiischemic activity compared to that of
BMS-180448 in the ischemic myocardium injury models of anesthetized
rats. As described above, the compounds of the present invention
exert excellent anti-ischemic activity both in vitro and in vivo,
while show low vasorelaxant activity, so that they can be used as
cardioprotectives for the prevention and treatment of myocardial
infarction, congestive heart failure, and stable angina.
The present invention includes pharmaceutical formulations used for
humans which are prepared in the customary manner by known methods,
for example by mixing the active ingredient or ingredients, such as
fillers, diluents, binders, humectants, disintegrants, etc.
Solid formulations for Oral administration are tablets, coated
tablets, dusting powders, granules, capsules, and pills, which can
contain more than one additives in addition to the active
ingredient or ingredients, for example starches, calcium carbonate,
sucrose, lactose, or gelatin. Besides simple additives, lubricants,
for example magnesium stearate and talc, can be used.
Liquid formulations for oral administration are suspension,
solution, emulsion, and syrup, which can contain the customary
excipients, for example, the liquid diluents such as water and
liquid paraffin, wetting agents, sweeteners, preservatives and
additives which improve the smell and taste.
Formulations for parenteral administration comprising sterile
solutions, suspensions, emulsions, lyophilized powders, and
suppositories, etc., can contain, in addition to the active
ingredient or ingredients, the customary water-insoluble excipients
and suspending agents, for example, propylene glycols, polyethylene
glycols, vegetable fats such as olive oil, and injectable esters.
Suppositories can contain the customary excipients, for example
witepsol, macrogol, tween 61, cacao fat, laurin fat, glycerol, or
gelatin, etc.
In general, it has proved advantageous in human medicine to
administer the active ingredient or ingredients according to the
present invention in total amounts of about 0.01 to about 1000,
preferably 0.1 to 500 mg/day based on adults with 70 Kg of body
weight, if appropriate in the form of several individual doses, to
achieve the desired results. However, it may be necessary to
deviate from the dosages mentioned, and in particular to do so as a
function of the nature and body weight of the object to be treated,
the nature and severity of the disease, the nature of the
formulation and of the administration of the medicament and the
period or interval within which administration takes place.
Thus in some cases it can suffice to manage with less than the
abovementioned amount of active ingredient, while in other cases
the abovementioned amount of active ingredient must be exceeded.
The particular optimum dosage and mode of administration required
for the active ingredient can be determined by any expert on the
basis of his expert knowledge.
The molecular structure of the compounds according to the present
invention was identified by IR spectroscopy, NMR spectroscopy, mass
spectroscopy, liquid chromatography, X-ray diffraction, optical
rotation analysis and elemental analysis.
PREPARATION EXAMPLES
Preparation Example
Preparation of Secondary Amines Including Imidazole Heterocycle
4-Chlorophenyl-1H-imidazol-2-ylmethylamine
The solution of 2-imidazolecarboxaldehyde (570 mg, 5.9 mmol) and
4-chloroaniline (756 mg, 5.9 mmol) in methanol (5 ml) was stirred
at 60.degree. C. for 4 hours, and allowed to cool to room
temperature. To the reaction was added NaBH.sub.4 (337 mg, 8.9
mmol), and the mixture was additionally stirred for an hour. Water
(20 mL) was added to the reaction, which was extracted with ethyl
acetate (50 mL). Organic layer was dried over MgSO.sub.4, filtered,
and concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (5% methanol in chloroform) to
give the title compound (660 mg, 53%).
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 4.27 (s, 3H), 5.40 (brs,
1H), 6.54 (m, 2H), 6.97 (m, 4H).
3-Chlorophenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 4.30 (s, 3H), 6.33-6.47
(m, H), 6.63-6.68 (m, 1H), 6.98 (m, 3H).
4-Methoxyphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 3.76 (s, 3H), 4.36 (s,
2H), 6.66-6.68 (m, 4H), 6.98 (s, 2H).
2-Methoxyphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 3.84 (s, 3H), 4.47 (d,
2H, J=4.6 Hz), 4.81 (brs, 1H), 6.52 (dd, 1H, J=8, 1.6 Hz),
6.67-6.86 (m, 3H), 6.98 (s, 2H).
4-Trifluoromethoxyphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 4.36 (s, 2H), 6.50 (dd,
2H, J=6.8, 2.2 Hz), 6.96-7.26 (m, 4H).
2-Trifluoromethoxyphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 4.47 (d, 2H, J=5.6 Hz),
4.72 (brs, 1H), 6.61-6.75 (m, 2H), 7.00 (s, 2H), 7.02-7.17 (m,
2H).
4-Trifluoromethylphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 4.31 (d, 2H, J=5.2 Hz),
6.36 (brs, 1H), 6.68 (d, 2H, J=8.8 Hz), 6.87 (s, 2H), 7.26 (d, 2H,
J=8.6 Hz).
2-Trifluoromethylphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 4.39 (d, 2H, J=5.4 Hz),
5.92 (brs, 1H), 6.68-7.00 (m, 4H), 7.35-7.45 (m, 2H).
4-Methylphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 2.18 (s, 3H), 4.30 (s,
2H), 6.56 (d, 2H, J=8.4 Hz), 6.88-6.96 (m, 4H).
4-Fluorophenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 4.31 (s, 2H), 6.45-6.61
(m, 2H), 6.71-6.95 (m, 4H).
4-Bromophenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 4.26 (s, 2H), 6.52 (brs,
1H), 6.53-6.58 (m, 2H), 6.87-6.89 (m, 2H), 7.10-7.16 (m, 2H).
2-Isopropylphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.23-1.27 (m, 6H), 4.36
(brs, 1H), 4.47 (s, 2H), 6.54-6.59 (m, 1H), 6.76-6.84 (m, 1H),
6.99-7.28 (m, 4H).
2,6-Dimethylphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 2.18 (s, 6H), 4.19 (s,
2H), 6.82-6.99 (m, 5H).
2,3-Dimethylphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 2.07 (s, 3H), 2.24 (s,
3H), 4.38 (s, 2H), 4.56 (brs, 1H), 6.46-6.56 (m, 2H), 6.87-6.95 (m,
3H).
2,4,6-Trimethylphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 2.16-2.27 (m, 9H), 4.19
(s, 2H), 6.82 (s, 2H), 7.00 (s, 2H).
4-Ethoxycarbonylphenyl-1H-imidazol-2-ylmethylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.28 (t, 3H), 4.20 (q,
2H), 4.33 (d, 2H, J=5.2 Hz), 6.54-6.63 (m, 2H), 6.88 (s, 2H), 7.68
(d, 2H, J=8.6 Hz).
1H-imidazol-2-ylmethylbenzylamine
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 3.71-3.83 (m, 4H),
6.90-6.97 (m, 2H), 7.03-7.24 (m, 5H).
Example 1
Preparation of
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
To the solution of the epoxide compound,
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (437 mg, 1.55 mmol) in acetonitrile (2 mL) was added
anhydrous CoCl.sub.2 (202 mg, 1.55 mmol). The reaction mixture was
stirred at 60.degree. C. for 10 h, then a saturated aqueous
solution of NaHCO.sub.3 (5 mL) was added to the mixture, which was
extracted with ethyl acetate (30 mL). The organic layer was washed
with brine, dried over anhydrous Na.sub.2SO.sub.4, and concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography (hexane:ethyl acetate=2:1) to yield the title
compound (304 mg, 40%).
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.49 (s, 3H), 3.60 (s,
3H), 3.63 (s, 3H), 4.32 (m, 1H), 4.57 (s, 1H), 5.14 (br s, 1H),
6.75 (br s, 2H), 6.97 (m, 4H), 7.27 (m, 2H), 7.93 (s, 1H), 8.08 (d,
1H).
Example 2
Preparation of
(2S,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (76 mg, 34%) was prepared using
(2S,3R,4R)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (129 mg, 0.46 mmol) and
4-chlorophenyl-1H-imidazol-2-ylmethylamine (95 mg, 0.46 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.66 (s, 3H), 3.60 (s,
3H), 3.69 (s, 3H), 3.87 (br s, 1H), 4.13 (m, 1H), 4.29 (d, 1H),
4.43 (d, 1H), 4.64 (s, 1H), 5.64 (d, 1H), 6.83 (d, 2H), 6.95 (m,
4H), 7.15 (d, 2H), 7.86 (s, 1H), 8.06 (m, 2H), 8.41 (s, 1H).
Example 3
Preparation of
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (76 mg, 34%) was prepared using
(2R,3R,4R)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (129 mg, 0.46 mmol) and
4-chlorophenyl-1H-imidazol-2-ylmethylamine (95 mg, 0.46 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.49 (s, 3H), 3.60 (s,
3H), 4.32 (m, 1H), 4.57 (s, 1H), 5.14 (br s, 1H), 6.75 (br s, 2H),
6.97 (m, 4H), 7.27 (m, 2H), 7.93 (s, 1H), 8.08 (d, 1H)<
Example 4
Preparation of
(2R,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (269 mg, 63%) was prepared using
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (129 mg, 0.46 mmol) and
4-chlorophenyl-1H-imidazol-2-ylmethylamine (183 mg, 0.88 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.66 (s, 3H), 3.60 (s,
3H), 3.69 (s, 3H), 3.87 (br s, 1H), 4.13 (m, 1H), 4.29 (d, 1H),
4.43 (d, 1H), 4.64 (s, 1H), 5.64 (d, 1H), 6.83 (d, 2H), 6.95 (m,
4H), 7.15 (d, 2H), 7.86 (s, 1H), 8.06 (m, 2H), 8.41 (s, 1H).
Example 5
Preparation of
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-trifluoromethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (146 mg, 22%) was prepared using
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (356 mg, 1.26 mmol) and
4-trifluoromethylphenyl-1H-imidazol-2-ylmethylamine (305 mg, 1.26
mmol), according to the same procedure used for the preparation of
example 0.1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.51 (s, 3H), 3.60 (s,
3H), 3.61 (s, 3H), 4.32 (m, 3H), 4.57 (s, 1H), 5.14 (br s, 1H),
6.85 (m, 2H), 6.95 (m, 4H), 7.38 (d, 2H), 7.91 (s, 1H), 8.05 (dd,
2H), 8.42 (m, 1H).
Example 6
Preparation of
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (280 mg, 28%) was prepared using
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (591 mg, 2.10 mmol) and
4-methoxyphenyl-1H-imidazol-2-ylmethylamine (427 mg, 2.10 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.47 (s, 3H), 3.59 (d,
6H), 3.68 (s, 3H), 4.30 (m, 2H), 4.54 (m, 2H), 5.02 (d, 1H),
6.67-6.78 (m, 4H), 6.89-7.26 (m, 3H), 8.04 (m, 2H).
Example 7
Preparation of
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyra-
n
The title compound (181 mg, 47%) was prepared using
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (200 mg, 0.71 mmol) and
4-trifluoromethoxyphenyl-1H-imidazol-2-ylmethylamine (183 mg, 0.71
mmol), according to the same procedure used for the preparation of
example 1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.50 (s, 3H), 3.60 (d,
6H), 4.2-4.50 (m, 2H), 4.58-5.65 (m, 2H), 5.18 (s, 1H), 6.91-6.95
(m, 7H), 8.00 (s, 1H), 8.05 (dd, 1H).
Example 8
Preparation of
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (310 mg, 41%) was prepared using
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (400 mg, 1.42 mmol) and
4-bromophenyl-1H-imidazol-2-ylmethylamine (359 mg, 1.42 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.48 (s, 3H), 3.61 (d,
6H), 4.10-4.19 (m, 2H), 4.20-4.40 (m, 2H), 5.13 (s, 1H), 6.70-7.01
(m, 6H), 7.21 (s, 1H), 7.94 (s, 1H), 8.06 (dd, 1H).
Example 9
Preparation of
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2,4-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (231 mg, 33%) was prepared using
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (400 mg, 1.42 mmol) and
2,4-dimethylphenyl-1H-imidazol-2-ylmethylamine (287 mg, 1.42 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.39 (s, 3H), 2.19 (s,
3H), 2.47 (s, 3H), 3.59 (d, 6H), 4.15-4.82 (m, 5H), 6.80-6.89 (m,
5H), 7.58 (d, 1H), 7.94-7.99 (dd, 1H), 8.62 (m, 1H).
Example 10
Preparation of
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2-isopropylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (140 mg, 20%) was prepared using
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (400 mg, 1.42 mmol) and
2-isopropylphenyl-1H-imidazol-2-ylmethylamine (306 mg, 1.42 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.22-1.29 (m, 10H), 3.60
(d, 6H), 4.07-4.63 (m, 5H), 6.79-7.35 (m, 6H), 7.78 (m, 1H), 7.99
(dd, 1H), 8.61 (m, 1H).
Example 11
Preparation of
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (253 mg, 37%) was prepared using
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (400 mg, 1.42 mmol) and
2,3-dimethylphenyl-1H-imidazol-2-ylmethylamine (287 mg, 1.42 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.39 (s, 3H), 2.17 (s,
3H), 2.41 (s, 3H), 3.61 (d, 6H), 4.26-4.74 (m, 5H), 6.76-6.95 (m,
4H), 6.98 (m, 1H), 7.58 (d, 1H), 7.95 (dd, 1H), 8.63 (d, 1H).
Example 12
Preparation of
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (416 mg, 49%) was prepared using
(2R,3R,4R)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (500 mg, 1.77 mmol) and
2,3-dimethylphenyl-1H-imidazol-2-ylmethylamine (358 mg, 1.77 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.39 (s, 3H), 2.17 (s,
3H), 2.41 (s, 3H), 3.61 (d, 6H), 4.26-4.74 (m, 5H), 6.76-6.95 (m,
4H), 6.98 (m, 1H), 7.58 (d, 1H), 7.95 (dd, 1H), 8.63 (d, 1H).
Example 13
Preparation of
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (570 mg, 60%) was prepared using
(2R,3R,4R)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (500 mg, 1.78 mmol) and
4-bromophenyl-1H-imidazol-2-ylmethylamine (450 mg, 1.78 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.48 (s, 3H), 3.61 (d,
6H), 4.10-4.19 (m, 2H), 4.20-4.40 (m, 2H), 5.13 (s, 1H), 6.70-7.01
(m, 6H), 7.21 (s, 1H), 7.94 (s, 1H), 8.06 (dd, 1H).
Example 14
Preparation of
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (446 mg, 86%) was prepared using
(2R,3R,4R)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (300 mg, 1.06 mmol) and
4-methoxyphenyl-1H-imidazol-2-ylmethylamine (216 mg, 1.06 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.47 (s, 3H), 3.59 (d,
6H), 3.68 (s, 3H), 4.30 (m, 2H), 4.54 (m, 2H), 5.02 (d, 1H),
6.67-6.78 (m, 4H), 6.89-7.26 (m, 3H), 8.04 (m, 2H).
Example 15
Preparation of
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-fluorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (650 mg, 48%) was prepared using
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (800 mg, 2.84 mmol) and
4-fluorophenyl-1H-imidazol-2-ylmethylamine (380 mg, 2.0 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.49 (s, 3H), 3.60 (d,
6H), 4.30 (m, 2H), 4.60 (m, 2H), 5.05 (m, 1H), 6.7.sup.6-6.97 (m,
7H), 7.95 (s, 1H), 8.03 (dd, 1H).
Example 16
Preparation of
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (500 mg, 58%) was prepared using
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (500 mg, 1.78 mmol) and
2-methoxyphenyl-1H-imidazol-2-ylmethylamine (253 mg, 1.25 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.38 (s, 3H), 3.60 (d,
6H), 3.91 (s, 3H), 3.97 (m, 1H), 4.74 (d, 1H), 4.60-4.84 (m, 3H),
6.80-7.03 (m, 6H), 7.58 (m, 1H), 7.99 (dd, 1H), 8.86 (m, 1H).
Example 17
Preparation of
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2-isopropylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (72 mg, 42%) was prepared using
(2R,3R,4R)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (100 mg, 0.35 mmol) and
2-isopropylphenyl-1H-imidazol-2-ylmethylamine (75 mg, 0.35 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.22-1.29 (m, 10H), 3.60
(d, 6H), 4.07-4.63 (m, 5H), 6.79-7.35 (m, 6H), 7.78 (m, 1H), 7.99
(dd, 1H), 8.61 (m, 1H).
Example 18
Preparation of
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(2-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (580 mg, 67%) was prepared using
(2R,3R,4R)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (500 mg, 1.78 mmol) and
2-methoxyphenyl-1H-imidazol-2-ylmethylamine (231 mg, 1.78 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.38 (s, 3H), 3.60 (d,
6H), 3.91 (s, 3H), 3.97 (m, 1H), 4.74 (d, 1H), 4.60-4.84 (m, 3H),
6.80-7.03 (m, 6H), 7.58 (m, 1H), 7.99 (dd, 1H), 8.86 (m, 1H).
Example 19
Preparation of
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(3-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (337 mg, 39%) was prepared using
(2R,3R,4R)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (500 mg, 1.77 mmol) and
3-chloroxyphenyl-1H-imidazol-2-ylmethylamine (366 mg, 1.77 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.51 (s, 3H), 3.61 (d,
6H), 4.20-4.57 (m, 2H), 4.57-4.59 (m, 2H), 5.17 (s, 1H), 6.69-6.73
(m, 3H), 6.94-7.01 (m, 4H), 7.89 (m, 1H), 8.04 (dd, 1H).
Example 20
Preparation of
(2S,3S,4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(3-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (280 mg, 35%) was prepared using
(2S,3S,4S)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (450 mg, 1.6 mmol) and
3-chloroxyphenyl-1H-imidazol-2-ylmethylamine (232 mg, 1.1 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.51 (s, 3H), 3.61 (d,
6H), 4.20-4.57 (m, 2H), 4.57-4.59 (m, 2H), 5.17 (s, 1H), 6.69-6.73
(m, 3H), 6.94-7.01 (m, 4H), 7.89 (m, 1H), 8.04 (dd, 1H).
Example 21
Preparation of
(2R,3R,4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-4-[N--
(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyra-
n
The title compound (155 mg, 40%) was prepared using
(2R,3R,4R)-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-6-nitro-2H-1--
benzopyran (200 mg, 0.71 mmol) and
4-trifluoromethoxyphenyl-1H-imidazol-2-ylmethylamine (183 mg, 0.71
mmol), according to the same procedure used for the preparation of
example 1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.49 (s, 3H), 3.60 (d,
6H), 4.20-4.50 (m, 2H), 4.58-5.65 (m, 2H), 5.18 (s, 1H), 6.91-6.95
(m, 7H), 7.99 (s, 1H), 8.04 (dd, 1H).
Example 22
Preparation of
(2S,3S,4R)-6-cyano-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (106 mg, 28%) was prepared using
(2S,3S,4S)-6-cyano-3,4-dihydro-2-dimethoxymethyl-3,4-epoxy-2-methyl-2H-1--
benzopyran (210 mg, 0.8 mmol) and
4-chlorophenyl-1H-imidazol-2-ylmethylamine (167 mg, 0.8 mmol),
according to the same procedure used for the preparation of example
1 above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.47 (s, 3H), 3.58 (s,
3H), 3.62 (s, 3H), 4.35 (m, 1H), 4.57 (s, 1H), 5.16 (br s, 1H),
6.81-6.93 (m, 3H), 7.17 (d, 1H), 7.38 (s, 1H), 7.51 (dd, 1H).
Example 23
Preparation of
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
To the solution of the nitro compound (521 mg, 1.07 mmol) prepared
from example 3 in methanol (3 mL) was added 10% Pd/C. The mixture
was hydrogenated at room temperature under 3 atmosphere pressure of
H.sub.2 for 12 hours, and filtered through a pad of Celite. The
filtrate was concentrated, and the residue was purified by silica
gel column chromatography (5% methanol in dichloromethane) to
afford the title compound (368 mg, 75%).
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.42 (s, 3H), 3.61 (s,
6H), 4.27 (m, 2H), 4.42 (s, 1H), 4.52 (d, 1H), 5.24 (m, 1H), 6.29
(s, 1H), 6.58 (d, 2H), 6.70 (d, 2H), 6.98 (m, 3H), 7.41 (m,
2H).
Example 24
Preparation of
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-chlorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
To the solution of the nitro compound (177 mg, 0.36 mmol) prepared
from example 1 in methanol (2 mL) was added a 0.4 M aqueous
solution of Cu(OAc).sub.2 (0.38 mL, 0.15 mmol), then slowly added
sodium borohydride (113 mg, 3.0 mmol) over 30 min. The reaction
mixture was stirred for an hour at room temperature, and ethyl
acetate (5 mL) was added to the reaction. The black precipitates
were removed by filtration, then to the filtrate was added a
saturated aqueous solution of NaHCO.sub.3 (5 mL). The mixture was
extracted with ethyl acetate (30 mL), and the organic layer was
washed with brine, dried over Na.sub.2SO.sub.4, filtered, and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (n-hexane:ethyl acetate=1:4) to
afford the title compound (58 mg, 35%).
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.42 (s, 3H), 3.61 (s,
6H), 4.27 (m, 2H), 4.52 (d, 1H), 4.42 (s, 1H), 5.24 (m, 1H), 6.29
(s, 1H), 6.58 (d, 2H), 6.70 (d, 2H), 6.98 (m, 3H), 7.41 (m,
2H).
Example 25
Preparation of
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-trifluoromethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (34 mg, 57%) was prepared using the nitro
compound (65 mg, 0.12 mmol) obtained from example 5, according to
the same procedure used for the preparation of example 24
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.38 (s, 3H), 3.60 (s,
3H), 4.06-4.85 (m, 3H), 4.41 (s, 1H), 5.06 (br s, 2H), 6.31 (s,
1H), 6.57 (d, 2H), 6.80-7.18 (m, 7H).
Example 26
Preparation of
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyra-
n
The title compound (23 mg, 24%) was prepared using the nitro
compound (100 mg, 0.19 mmol) obtained from example 21, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.50 (s, 3H), 3.60 (d,
6H), 4.20-4.50 (m, 2H), 4.59 (s, 2H), 5.18 (s, 1H), 6.30 (s, 1H),
6.60 (dd, 2H), 6.70-6.96 (m, 6H).
Example 27
Preparation of
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (19 mg, 15%) was prepared using the nitro
compound (135 mg, 0.28 mmol) obtained from example 12, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.29 (s, 3H), 2.27 (s,
3H), 2.43 (s, 3H), 3.60 (s, 6H), 4.41-4.63 (m, 5H), 6.57 (dd, 1H),
6.70-7.19 (m, 6H), 7.40 (d, 1H).
Example 28
Preparation of
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (21 mg, 23%) was prepared using the nitro
compound (100 mg, 0.21 mmol) obtained from example 14, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.36 (s, 3H), 3.60 (d,
6H), 3.64 (s, 3H), 4.20-4.60 (m, 3H), 4.45 (s, 1H), 4.70-4.90 (m,
2H), 6.50 (m, 1H), 6.70 (dd, 1H), 6.80-7.00 (m, 6H), 7.40 (d,
1H).
Example 29
Preparation of
(2R,3R,4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (50 mg, 53%) was prepared using the nitro
compound (100 mg, 0.19 mmol) obtained from example 13, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.48 (s, 3H), 3.61 (d,
6H), 4.10-4.19 (m, 2H), 4.22 (s, 2H), 5.13 (s, 1H), 6.33-7.15 (m,
9H).
Example 30
Preparation of
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(2,3-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (35 mg, 54%) was prepared using the nitro
compound (70 mg, 0.14 mmol) obtained from example 11, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.29 (s, 3H), 2.27 (s,
3H), 2.43 (s, 3H), 3.60 (s, 6H), 4.41-4.63 (m, 5H), 6.57 (dd, 1H),
6.70-7.19 (m, 6H), 7.40 (d, 1H).
Example 31
Preparation of
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(2-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (74 mg, 66%) was prepared using the nitro
compound (80 mg, 0.16 mmol) obtained from example 16, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.30 (s, 3H), 3.60 (d,
6H), 3.80 (s, 3H), 4.10-4.30 (m, 2H), 4.45 (s, 1H), 4.70-4.90 (m,
2H), 6.50 (dd, 1H), 6.70-7.00 (m, 7H), 7.40 (d, 1H).
Example 32
Preparation of
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-methoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (74 mg, 77%) was prepared using the nitro
compound (103 mg, 0.21 mmol) obtained from example 6, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.36 (s, 3H), 3.60 (d,
6H), 3.64 (s, 3H), 4.20-4.60 (m, 3H), 4.45 (s, 1H), 4.70-4.90 (m,
2H), 6.50 (m, 1H), 6.70 (dd, 1H), 6.80-7.00 (m, 6H), 7.40 (d,
1H).
Example 33
Preparation of
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(2,4-dimethylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (54 mg, 67%) was prepared using the nitro
compound (86 mg, 0.18 mmol) obtained from example 9, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.26 (s, 3H), 2.20 (s,
3H), 2.43 (s, 3H), 3.58 (s, 6H), 4.36-4.54 (m, 3H), 4.60 (m, 2H),
6.56 (dd, 1H), 6.70 (dd, 1H), 6.80-7.15 (m, 6H), 7.36 (d, 1H).
Example 34
Preparation of
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(2-isopropylphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (30 mg, 73%) was prepared using the nitro
compound (45 mg, 0.09 mmol) obtained from example 10, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.22-1.29 (m, 9H), 3.60
(d, 6H), 4.10-4.62 (m, 5H), 6.50-6.77 (m, 2H), 6.85-7.30 (m, 6H),
7.60 (m, 1H).
Example 35
Preparation of
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-trifluoromethoxyphenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyra-
n
The title compound (34 mg, 72%) was prepared using the nitro
compound (50 mg, 0.10 mmol) obtained from example 7, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.50 (s, 3H), 3.60 (d,
6H), 4.20-4.50 (m, 2H), 4.59 (s, 2H), 5.18 (s, 1H), 6.30 (s, 1H),
6.60 (dd, 2H), 6.70-6.96 (m, 6H).
Example 36
Preparation of
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[N--
(4-bromophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (41 mg, 88%) was prepared using the nitro
compound (50 mg, 0.10 mmol) obtained from example 8, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.48 (s, 3H), 3.61 (d,
6H), 4.10-4.19 (m, 2H), 4.22 (s, 2H), 5.13 (s, 1H), 6.33-7.15 (m,
9H).
Example 37
Preparation of
(2S,3S,4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-4-[(N-
-(4-fluorophenyl)-N-(1H-imidazol-2-ylmethyl)amino]-2H-1-benzopyran
The title compound (44 mg, 95%) was prepared using the nitro
compound (50 mg, 0.10 mmol) obtained from example 15, according to
the same procedure used for the preparation of example 23
above.
.sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 1.49 (s, 3H), 3.60 (d,
6H), 4.30 (m, 4H), 4.98 (s, 1H), 6.33 (s, 1H), 6.55 (dd, 2H),
6.60-6.92 (m, 6H).
EXPERIMENTAL EXAMPLES
The following experiments were made on the compounds of the formula
1 to investigate their pharmacological actions.
Experimental Example 1
Inhibitory Effects on HUVEC Tube Formation
The Inhibitory effects of the compounds of the formula 1 on
angiogenesis were meassured by the vascular tube formation assay of
HUVECs (Human umbilical vein endothelial cells), which is one of
the major angiogenic steps.
HUVECs were isolated from human umbilical vein, and cultured.
HUVECs within passage 5 from confluent cultures were detached, and
plated onto a layer of a bFGF (basic fibroblast growth
factor)-reduced and polymerized Matrigel. Matrigel cultures were
incubated with or without the compounds of formula 1, and the
changes of cell morphology were captured through a phase contrast
microscope and photographed.
The effects on tube formation of the test compounds were compared
with the vehicle treated controls, then confirmed their in vitro
anti-angiogenic effect indirectly. The results are given in table
1.
TABLE-US-00001 TABLE 1 Inhibitory Effects on HUVEC Tube Formation
Tube formation Example 10 .mu.M 50 .mu.M 100 .mu.M Example 1 + + ++
Example 2 ++ +++ Example 3 ++ Example 4 + Example 5 ++ Example 6 +
Example 7 + Example 8 + Example 9 + Example 10 ++ Example 12 +
Example 13 + Example 14 + Example 22 + ++ Example 23 +++ Example 24
+ + Example 25 +/- +/- Example 26 + Example 27 +/- Example 28 +
Example 29 + -; no inhibition, +/-; week inhibition, +; moderate
inhibition, ++; strong inhibition +++; complete inhibition
As seen in table 1, the compound of example 2 showed potent
inhibitory effects on HUVEC tube formation in a dose-dependent
manner with strong inhibition at 10 uM, and complete inhibition at
100 uM. The compounds of example 3, 5, and 10 strongly inhibited
HUVEC tube formation at 50 uM, and in particular, the compound of
example 23 didn't show any tube like structures at 50 uM. The
compounds of the present invention demonstrated antiangiogenic
actions by inhibiting vascular tube formation, one of major
angiogenic steps.
With such an excellent suppressive activity against angiogenesis,
the compounds of the present invention can be usefully applied for
the treatment of various diseases induced by angiogenesis, such as
cancers, rheumatoid arthritis, diabetic retinopathy, psoriasis, and
AIDS complications, etc.
Experimental Example 2
In Vivo Mouse Matrigel Plug Assay
Antiangiogenic activities in vivo of the compounds of the formula 1
were determined by mouse matrigel plug assay.
The mixture of Matrigel, heparin (30 units), and bFGF (25 ng) were
injected subcutaneously into C57BL/6 mice. The test compounds were
injected subcutaneously at 2.2 mg/mL with matrigel, or
administrated orally at 2 mg/day (twice a day, each 1 mg) during 4
days (total 8 mg/mouse). After 4-7 days, the skin of the mouse was
pulled back, and matrigel plug was excised. The hemoglobin contents
inside the Matrigel plugs were measured using the Drabkin method
and Drabkin reagent (kit 525, Sigma) for the quantitation of blood
vessel formation. The results are given in table 2.
TABLE-US-00002 TABLE 2 In vivo mouse matrigel plug assay %
Inhibition S. C oral control 0 0 Example 1 79 94
As shown in table 2, the compound of example 1 markedly inhibited
the hemoglobin quantity to 79%, or 94% each, by subcutaneous
injection (2.2 mg/mL) or oral administration (8.8 mg/mouse), which
demonstrated the antiangiogenic activity in vivo.
With such an excellent suppressive in vivo activity against
angiogenesis, the compounds of the present invention can be
usefully applied for the treatment of various diseases induced by
angiogenesis, such as cancers, rheumatoid arthritis, diabetic
retinopathy, psoriasis, and AIDS complications, etc.
Experimental Example 3
CAM (Chorioallatonic Membrane) Assay
Chick chorioallantoic membrane (CAM) assays were performed to
measure the inhibitory effects on angiogenesis in vivo of the
compounds of the formula 1.
Fertilized chick eggs were incubated under constant humidified
(90%) egg breeder at 37.degree. C. On the third day of incubation,
about 2 mL of egg albumin was aspirated by an 18-gauge hypodermic
needle through the small hole drilled at the narrow end of the
eggs, to detach the developing CAM from the shell. And the shell
covering the air sac was punched out and removed by forcep, and the
shell membrane on the floor of the air sac was peeled away. After
more incubation of two more days, sample-loaded thermanox
coverslips were air-dried, and applied to the CAM surface to test
the angiogenic inhibition by the test compounds. Three days later,
1 to 2 mL of 10% fat emulsion (Intralipose) was injected into the
chorioallantoic and observed avascular zone under a dissecting
microscope. The negative control was implanted only thermanox
coverslip, while the positive control was treated with retinoic
acid (1 .mu.g/egg). The response that CAM showed similar avascular
zone to that treated with retinoic acid was scored as positive, and
the percentage of positive eggs to total numbers of eggs tested was
calculated (%). Independent experiment was repeated three times and
at least more than 20 eggs in each experiment were used.
TABLE-US-00003 TABLE 3 Anti-angiogenic activity in CAM assay %
Inhibition 0.5 .mu.M/egg 1.5 .mu.g/egg Example 1 62 84
The negative control implanted only thermanox coverslip formed new
branches from the existing vessels, demonstrating normal vascular
development similar to that observed with untreated CAM. The
positive control treated with retinoic acid significantly inhibited
microvessel formation, especially larger vessels formation. The
compound of example 1 showed 62% and 84% inhibition at the
concentration of 0.5 .mu.g/egg, and 1.5 .mu.g/egg, each. The
positive responses by the compound of example 1 was significant,
and the inhibitory effect on chicken embryonic angiogenesis was
dose-dependent.
With such an excellent anti-angiogenic activities on in vivo CAM
assay, the compounds of the present invention can be usefully
applied for the treatment of various diseases induced by
angiogenesis, such as cancers, rheumatoid arthritis, diabetic
retinopathy, psoriasis, and AIDS complications, etc.
Experimental Example 4
Human Tumor Xenografts in Nude Mice
Human tumor xenograft experiments were performed to evaluate
whether the compounds of the formula 1 inhibit growth of human
tumors implanted in nude mice.
Nude mice (BALB/c nu/nu, male) were purchased from Charls River
Japan, Inc., and housed and treated under SPF (Special Pathogen
Free) facilities according to the regulation of NIH (national
institutes of health). A549 cells isolated from human non small
cell lung cancer (NSCLC) were purchased from ATCC (American tissue
cancer collection, USA), and maintained as an exponentially growing
monolayer in Korea Research Institute of Chemical Technology.
Effects of the compounds of present invention on implanted tumor
growth were measured by the comparison of tumor sizes, in addition,
the changes of body weights and survival % of nude mice were
observed.
(1) Inhibition on A549 NSCLC Growth
Nude mice had been adjusted to the laboratory for 2-3 weeks after
purchase, and all procedures were performed on male mice of 8 weeks
of age, within a weight range of 18-20 g.
A549 tumor xenografts were established in the right flank of mice
by subcutaneous injection of cells in 3.times.3.times.3 mm.sup.3
size of culture. 24 hours after cell implantation, the
administration of the compounds was started and which day is
defined as day 1. Nude mice were injected intraperitoneally with
the compound of example 23 (50 mg/kg) or vehicle (PBS containing
0.5% tween 80) once daily from day 1 to day 20. Tumor volume (V)
was assessed by caliper measurement using the mathematical formula
1, where a was the longest diameter across the tumor and b was the
corresponding perpendicular short diameter. Volume
(mm.sup.3)=a.times.b.sup.2/2 [Mathematical Formula 1]
Inhibition (%) was calculated as
(1-(V.sub.T).sub.n/-(V.sub.C).sub.n).times.100, where
(V.sub.T).sub.n and (V.sub.C).sub.n were the mean tumor volume of
treated and control group at n days after administration of the
compound. Each group consisted of 8 mice, and statistical
significance was represented as student-T test (* p<0.05).
TABLE-US-00004 TABLE 4 Inhibition of the compounds on A549 NSCLC
growth in nude mice xenografts Tumor volume (mm.sup.3), Inhibition
(%) 25 days 35 days 45 days 65 days Control 152.9 308.5 483.9
1034.9 Example 23 92.2 186.8 244.3* 494.8* (50 mg/Kg) (40%) (39%)
(50%) (52%) (Inhibition %)
As represented in table 4, the compound of example 23 significantly
inhibited A549 NSCLC growth from 45 days to 61 days after
implantation by 50-52%. With such an excellent anti-cancer activity
in vivo human tumor xenografts experiment as well as
anti-angiogenic properties, the compounds of the present invention
can be usefully applied as anti-cancer agents.
(2) Effects on Changes of Body Weights
The effects of the compounds of the formula 1 on changes of body
weights were determined. Body weights of nude mice were measured
using AND balance at 5 day intervals starting from the date of
administration of compounds, and represented as a mean value of
treated group (W.sub.C) and control group (W.sub.T),
respectively.
TABLE-US-00005 TABLE 5 Effects of the compounds of formula 1 on
changes of body weight in nude mice implanted A549 Body weight (g)
1 day 14 days 25 days 35 days Control 22.50 25.16 25.43 25.88
Example 23 22.30 25.42 26.17 27.10 (50 mg/Kg)
As shown in table 5, retardation or loss of body weight gain was
not observed by the treatment of the compound of example 23, then
the compounds of present invention cab be used as anti-cancer
agents without side effects of retardation or loss of weight
gain.
(3) Effects on Survival Percents of Mice
Survival percents of mice were measured to determine the toxicity
by administration of the compounds of the formula 1, and to
demonstrate the survival rate of A549 implanted mice as time
passed. Survival percents were represented using following
mathematical formula 2, where N.sub.0 is the number of mice at the
first day of administration, and N.sub.n is the number of mice at n
days after administration. Survival %=N.sub.n/N.sub.0.times.100
[Mathematical formula 2]
TABLE-US-00006 TABLE 6 Effects on survival % of the compounds in
A549 implanted mice Survival % 1 day 5 days 15 days 20 days Control
100% 100% 100% 100% Example 23 100% 100% 100% 100% (50 mg/Kg)
As seen in table 6, the compound of example 23 showed 100% of
survival rate, which demonstrates the significantly reduced side
effects and toxicities compared to the traditional cytotoxic
anti-cancer agents. As described above the compounds of present
invention show the excellent inhibition on tumor growth with
significantly reduced toxicities, therefore the compounds of
present invention can be usefully applied as anti-cancer
agents.
Experimental Example 5
Protective Activities Against Iron-Induced Neuronal Damage
In order to examine whether the compounds of the formula 1 suppress
the iron-induced neuronal damage and death, experiments were
conducted as follows.
From the brains of 17-18 day-old rat embryos, cerebral cortical
neurons were isolated and then, cultured at 37.degree. C. for 7-9
days in a 5% CO.sub.2 incubator. The cortical cell cultures were
washed twice with a minimum essential medium (MEM) to reduce the
serum concentration to 0.2% and pre-treated with test compounds at
30, 7.5, 1.875, and 0.469 .mu.M of final concentrations, each for
30 min. The test compounds were dissolved in DMSO and diluted in a
medium. At this time, the final concentration of DMSO was not
allowed to exceed 0.2%. For a control group, only vehicle was
applied.
After the pre-treatment with test compounds or vehicle, FeSO.sub.4
was added to a final concentration of 50 .mu.M, and the cultures
were maintained for 24 hours in a CO.sub.2 incubator. During
incubation, lactate dehydrogenase (LDH) was released into the
medium upon neuronal death by the oxidative toxicity of iron. The
extent of neuronal damage was assessed by measuring the amount of
LDH released into the media. The protective effect of the compounds
of interest on neurons was evaluated by calculating the reduction
rate of released LDH of treated group compared with that of the
control group, and IC.sub.50 was calculated as the least linear
regression analysis of dose-response curve. The results are given
in Table 7, below.
TABLE-US-00007 TABLE 7 Protective Effect of Compounds of Formula 1
on Neurons Neuroprotection inhibition % Compound (30 .mu.M)
IC.sub.50 (.mu.M) Example 2 92% 6.2
As seen in Table 7, the compound of example 2 showed 92% of
inhibition on LDH release with the IC.sub.50 of 6.2 .mu.M, which
demonstrates that the compound has very potent protective activity
against the iron-included damage to neurons.
Since the compounds of the present invention showed an excellent
protective effects on neurons, they can be used as preventive or
curative agents for the medical treatment of the neurological
disorders caused by the damage or death of neurons, such as
cerebral stroke and dementia as well as for the medical treatment
of inflammatory diseases such as arthritis, cardiac infarction, and
acute/chronic tissue damage.
Experimental Example 6
Inhibitory Activity Against Iron-induced Lipid Peroxidation
In order to examine whether the compounds of the formula 1 suppress
the iron-induced lipid peroxidation, experiments were conducted as
follows.
The rat brain was homogenized in a Krebs buffer (15 mM HEPES, 10 mM
glucose, 140 mM NaCl, 3.6 mM KCl, 1.5 mM CaCl.sub.2, 1.4 mM
KH.sub.2PO.sub.4, 0.7 mM MgCl.sub.2, pH 7.4) and the supernatant
separated by centrifugation at 12,000 rpm for 10 min was used for
further experiments. FeCl.sub.2 was added to a final concentration
of 400 .mu.m in the brain homogenate which was then allowed to
stand at 37.degree. C. for 30 min. for the facilitation of
oxidation. Each of the test compounds was added at a concentration
of 30 .mu.M and vehicle was used as a control.
Iron facilitates the oxidation of the brain homogenate to produce
malondialdehyde (MDA), a lipid peroxidation product. Thus, the
lipid peroxidation was determined by MDA quantification. The
inhibitory effect of the test compounds against the lipid
peroxidation was evaluated by calculating MDA reduction rate of the
test compounds compared with that of the control group.
Typically, the MDA quantification is achieved by reacting samples
with 2-thiobarbituric acid (TBA) and measuring the absorbance at
530 nm. However, this method is unsuitable to treat samples on a
large scale because of a boiling step. Thus, in this experiment,
N-methyl-2-phenylindole was used instead of TBA. In this case, one
molecule of MDA reacts with two molecules of
N-methyl-2-phenylindole to form a chromogen which shows a maximal
absorbance at 586 nm and requires no boiling steps. Bioxytech.sup.R
LPO-586 Kit was used for MDA quantification. The results are given
in Table 8, below.
TABLE-US-00008 TABLE 8 Inhibitory Effect of Compounds of Formula 1
on Lipid Peroxidation by iron Compounds Concentration (.mu.M) %
Inhibition Example 24 30 83 Example 25 30 97
As seen in Table 8, the compounds of the present invention suppress
the iron-induced lipid peroxidation. In particular, the compounds
of Examples 24 and 25 showed very potent inhibitory activity
against the iron-induced lipid peroxidation with inhibitory effects
of 83% and 97%, respectively at the concentration of 30 .mu.M.
With excellent inhibitory activity against lipid peroxidation, the
compounds of the present invention can be used for the prevention
and treatment of neurodegenerative diseases such as cerebral stroke
and dementia, inflammatory diseases such as arthritis, cardiac
infarction, and acute/chronic tissue damage, which may be caused by
the lipid peroxidation and its accumulation in tissues.
Experimental Example 7
Vasorelaxation Effects on Isolated Blood Vessels of Rats
The following experiment was conducted to examine whether the
compounds of the formula 1 have vasorelaxation effects.
Male Sprague-Dawly rats (350-450 g, obtained from the Experimental
Animal Team of the Korea Research Institute of Chemical Technology)
were knocked unconscious by hitting the occipital region,
sacrificed by cervical dislocation, and underwent thoracotomy.
After being quickly removed, the thoracic aorta was deprived of the
adipose tissue and cut into aortic rings of 3 mm width. The aorta
was lightly rubbed with a modified Krebs Henseleit buffer
(Physiological Salt Solution, PSS) soaked cotton club to remove the
inner epithelial layer therefrom. While being suspended in an organ
bath containing a physiological buffer, the vascular tissue was
allowed to equilibrate under a resting tension of 2 g and then,
stand for 1 hour at 37.degree. C. for stabilization with a supply
of a carbogen consisting of 95% O.sub.2-5% CO.sub.2.
Thereafter, the vascular tissue was constricted with 10.sup.-5 M
phenylephrine and washed several times with PSS and this procedure
was repeated again to ensure the stable reactivity of vascular
smooth muscle to repetitive constriction/dilatation.
In addition, 3.times.10.sup.-6 M methoxamine was used to induce an
intensive constriction in the vascular smooth muscle. When the
vasoconstriction induced by the methoxamine reached and maintained
a maximum, test compounds and controls were cumulatively added to
the organ baths in concentrations of 1, 3, 10 and 30 .mu.M so as to
induce vasodilatation. Cromakalim, BMS-180448, and BMS-191095 were
used as the controls.
Following the addition of test compounds, the change in the maximal
constriction induced by methoxamine was calculated to plot a
concentration-relaxation response curve. Through a linear
regression analysis, IC.sub.50, the drug concentration at which the
vascular tissue is 50% dilated, was obtained for each compound. The
results are given in Table 9, below.
TABLE-US-00009 TABLE 9 Vasorelaxation effects of the Compounds of
formula 1 on Methoxamine Induced Vasoconstriction Compounds
Vasorelaxation Effects (IC.sub.50, .mu.M) Cromakalim 0.067
BMS-180448 1.38 BMS-191095 2.14 Example 1 9.83
Cromakalim showed a potent vasorelaxation effect with 0.067 .mu.M
of IC.sub.50 on the isolated rat aorta constricted with methoxamine
(3 .mu.M), while IC.sub.50 of BMS-180448 and BMS-191095 were 1.38,
and 2.14 .mu.M respectively, showing vasodrelaxation potencies
twenty times and thirty times as weak as Cromakalim. On the other
hand, the compound of example 1 represented 9.83 .mu.M of
IC.sub.50, so that its vasorelaxation effect was significantly
weaker than the controls, Cromakalim, BMS-180448, and
BMS-191095.
When exerting their actions on the KATP present in the heart, the
compounds according to the present invention play a role in
protecting the heart. On the other hand, the KATP openers acting on
the KATP present in peripheral vascular smooth muscle dilate the
blood vessels, lowering the blood pressure. Hypotension may mask
any cardioprotective effects due to reduction in coronary artery
perfusion pressure, and would limit utility in treating myocardial
ischemia. Therefore, the compounds of the present invention may be
more optimal for cardioprotectives by virtue of their weak
vasodilatation activity.
As illustrated above, the compounds of the present invention are so
low in the vasorelaxant potencies that they are improved in the
selectivity for heart protective function.
Experiment Example 8
Cardioprotective Activity in Isolated Ischemic Heart Models of
Rats
In order to determine whether the compounds of the chemical formula
1 are protective for ischemic hearts in vitro, experiments
determining the anti-ischemic effects of the compounds on isolated
rat hearts were conducted as follows.
For all in vitro studies, isolated rat hearts were used according
to the published methods after some modification [HJ Ring,
Arzneim.-Forsch./Drug Res. 39 (II), 1535 (1989): T. Krzeminski, et
al., J. Pharmacological Methods, 25, 95, (1991)]
Male Sprague-Dawley rats weighing 300-450 g were anesthetized with
sodium pentobarbital (100 mg/kg, i.p.). The tail vein was injected
with heparin (1,000 U/kg) and then the trachea was intubated. While
rats were mechanically ventilated with a rodent ventilator (Model
7025, Ugobasile, Italy), their hearts were perfused in situ with
oxygenated modified Krebs-Henseleit bicarbonate buffer (described
herein) by retrograde aortic cannulation. The hearts were then
excised and moved to a Langendorff apparatus (H.S.E., Germany),
where they were perfused with oxygenated modified Krebs-Henseleit
bicarbonate buffer containing (in mM) NaCl 116, NaHCO.sub.3 24.9,
KCl 4.7, MgSO.sub.4 1.1, KH.sub.2PO.sub.4 1.17, CaCl.sub.2 2.52,
glucose 8.32 and pyruvate 2.0 at a constant perfusion pressure (85
mm Hg). A latex balloon filled with solvent (ethanol:water=1:1
(v/v)) and attached to a metal cannula was placed in the left
ventricle through pulmonary vein and connected to a Isotec pressure
transducer (H.S.E., Germany) for measurement of left ventricular
pressure (LVP). The hearts were allowed to equilibrate for 15 min,
at which time left ventricular end-diastolic pressure (EDP) was
adjusted to 5 mm Hg and this balloon volume was maintained
throughout the experiment. Then, baseline contractile function,
heart rate (HR), and coronary flow (CF) (extracorporeal
electromagnetic flow probe, Narco Bio-System, U.S.A.) were
measured. Cardiac contractile function was calculated by
subtracting LVEDP from LV peak systolic pressure (LVSP), yielding
developed pressure (LVDP). Double product (DP), another important
parameter for assessing cardiac performance, was calculated by
multiplying HR by LVDP. Throughout the experiment, all these
parameters were measured, and calculated before and 10 min after
pretreatment with each compound and 30 min after the onset of
reperfusion with buffer. Data on reperfusion DP were further
expressed as the percentage to pretreatment DP.
After stabilization for 15 min, the hearts were pretreated for 10
min with respective drugs (10 .mu.M, 0.04% DMSO) or vehicle (0.04%
DMSO) before onset of global ischemia; test agents were
administered directly into the oxygenator of the Langendorff
apparatus immediately above the aortic root in a retrograde fashion
as solutions in the perfusate. We then rendered the hearts globally
ischemic by completely shutting off the perfusate for 30 min.
Severity of ischemia was determined as the time to contracture
(TTC, min) during global ischemia in which the first 5 mmHg
increase in EDP was observed. Then, the hearts were reperfused and,
30 min later, contractile function (LVDP, DP) and cumulative
reperfusion lactate dehydrogenase (LDH) release were measured. LDH
was measured as a sensitive index for loss of cell viability with a
kit supplied by Boerhinger Mannheim based on the technique of
Wroblewski and LaDue [F. Wroblewski and J S. La Due, Proc Soc Exp
Biol Med 90, 210, (1955)].
TABLE-US-00010 TABLE 10 Cardioprotective Effect of Compounds of
Formula 1 Cardioprotection on Ischemic heart (.mu.M) TTC Test Drugs
LDVP .times. HR (%) EDP (MmHg) (min) LDH (U/g) Vehicle 23.0 43.4
20.3 29.9 BMS-180448 67.6 16.5 27.8 17.2 Example 1 55.7 24.0 28.0
10.7
In vehicle-treated group, reperfusion DP (LVDP.times.HR), a index
for contractility function, was decreased to 23.0% of pretreatment
DP, and EDP was increased to 43.3 mmHg from 5 mmHg, and TTC was
20.3 min, and reperfusion LDH release was 29.9 U/g as shown in the
above table 10. In BMS-190448 treated group, reperfusion
contractile function (DP, LVDP.times.HR) was 67.6% of pretreatment
DP, which was significantly improved compared to vehicle treated
group. EDP was 16.5 mmHg, significantly lower than control, and TTC
was 27.8 min, prolonged than control, and reperfusion LDH release
was 17.2 U/g, decreased than control. Then, in BMS-180448 treated
group all parameters showed significant protective effect on
ischemic heart. The compound of example 1 showed a good
cardioprotective effect similar to BMS-180448, of which contractile
function (LVDP.times.HR) was improved to 55.7% of pretreatment
index, and EDP was 24.0 mmHg, and TTC was 28 min, and reperfusion
LDH release was 10.7 U/g. However, because the compound of example
1 is 7 times lower vasorelaxant potency (IC.sub.50=9.83 .mu.M) than
BMS-180448 does (IC.sub.50=1.38 .mu.M), it is superior to
BMS-190448 in cardioselective antiischemic activity lower
vasodilation potency than BMS-180448 (IC.sub.50=1.38 .mu.M).
Consequently, the compounds of the present invention can be used
for the treatment of ischemic heart diseases by virtue of their
excellent selectivity and protective activity against ischemic
cardiovascular diseases such as myocardial infarction, heart
failure, and angina pectoris, etc.
Experiment Example 9
Cardioprotective Activity in Ischemic Heart Models of Rats
In order to determine whether the compounds of formula 1 are
protective for ischemic hearts, experiments determining the
anti-ischemic effects of the compounds on rats were conducted as
follows.
Male rats (350-450 g, obtained from the Experimental Animal Team of
the Korea Research Institute of Chemical Technology) were
anesthetized by the intraperitoneal injection of pentobarbital at a
dose of 75 mg/kg. After trachetomy, the rats were rendered to
respire artificially at a rate of 60/min with a stroke volume of 10
ml/kg. Cannulars were inserted into the fermoral vein and the
fermoral artery and used for drug administration and blood pressure
measurement, respectively.
In the ischemic myocardial injury models, the body temperature has
an important influence on the results. To avoid the change in the
body temperature, a body temperature measuring probe was inserted
into the rectum of each rat and the body temperature was constantly
kept at 37.degree. C. with the aid of a homeothermic blanket
control unit.
Afterwards, during testing, a continuous measurement was made of
the mean arterial blood pressures and heart rates from the rats.
For the measurement of the blood pressure, a pressure transducer
(Statham P23XL, Grass Ins., MA, U.S.A.) was used. The heart rate
was measured by a tachometer (ECG/RATE Coupler, Hugo Sachs
Electronic, German) identified as Biotachometer. In addition, all
of the changes occurring were continuously recorded through the
Gould 2000 chart recorder (Graphtech Linearcorder W R 3310, Hugo
Sachs Electronic).
The left coronary artery was occluded according to the Selye H.
method as follows. The rats underwent a left thoracotomy operation
for partial opening of the chest and the right-side chest was
pressurized by the middle finger of the left hand to push the heart
out. Immediately after the left anterior descending coronary artery
hereinafter referred to as (LAD) was carefully stitched using a
suture needle with 5-0 silk ligature, the heart was then
repositioned in the thoracic cavity while both ends of the ligature
were situated outside. The opposite ligature ends were passed
through a PE tube (PE100, 2.5 cm) and allowed to stand loose for 20
min for stabilization. Via the cannula inserted into the femoral
vein, vehicles or drugs were administered into the rats which were
rendered to stand for 30 min in order to sufficiently elicit the
efficacies of the drugs. BMS-180448 was used as a control drug and
the i.v. administration dose was 0.3 mg/kg for all test drugs of
interest and the control drug.
Next, the PE tube which had the doubled strands of the ligature
passed therethrough was pushed toward the heart and then, set
upright by tightly pulling end regions of the ligature with a
hemostatic pincette while pressing the coronary artery. The PE tube
was allowed to stand for 45 min for the occlusion of the coronary
artery, followed by the removal of the hemostatic pincette and
then, by the reperfusion for 90 min.
After the reocclusion of the coronary artery in accordance with the
above procedure, the rats were administered with 2 ml of a 1% Evans
blue through an intravenous route. Subsequently, an excess of
pentobarbital was intravenously injected to kill the rats, after
which the heat was removed and then, deprived of the right
ventricle and both atria. The left ventricle was cut horizontally
to the heart apex into 5 or 6 slices which were weighed. From the
surface of each slice, images were input with the aid of a Hi-scope
into a computer installed with an image analyzing program (Image
Pro Plus). From the images input into the computer, the area of the
normal blood stream tissue region which appeared blue in a computer
monitor and the area which appeared colorless were measured. The
percentage of the colorless area to the total area of each slice
was calculated and multiplied by the weight of each slice to
determine the area at risk (AAR) of each slice. The AAR obtained
from each slice was summed for all slices and the total AAR was
divided by the total weight of the left ventricle to yield % AAR,
as shown in the following mathematical formula 3: AAR (%)=(summed
AAR for all slices)/(total weight of left ventricle).times.100
[Mathematical Formula 3]
In addition, the heart slices were incubated for 15 min in
2,3,5-triphenyltetrazolium chloride (TTC) phosphate buffer (pH 7.4)
at 37.degree. C. and fixed for 20-24 hours in a 10% formalin
solution. During this fixation, 2,3,5-triphenyltetrazolium chloride
was reduced into formazan dye by the myocardial dehydrogenase and
its cofactor NADH, so that the normal regions of the tissue were
colored brick-red. In contrast, the infarct zones of the tissue
were deficient in the dehydrogenase and its cofactor, so that no
reduction occurred on the 2,3,5-triphenyltetrazolium, allowing the
color to remain unchanged.
According to whether the tissue regions were colored by
2,3,5-triphenyltetrazolium, a measurement was made of the areas of
the normal and infarct zones in each ventricle slice. The infarct
zone area of each slice was summed for all slices and the resulting
summed infarct zone area was divided by total AAR weight or total
left ventricle weight to yield % IZ as shown in the following
mathematical formula 4: IZ (%)=(summed infarct zone area)/(total
left ventricle area).times.100 [Mathematical Formula 4]
TABLE-US-00011 TABLE 11 Anti-Ischemic Effect of Compounds of
Formula 1 Anti-ischemic effect Rat in vivo (0.3 mg/kg) Compounds
AAR/LV (%) IZ/AAR (%) Vehicle 39.8 60.8 BMS-180448 38.8 39.1
Example 1 33.4 41.2
In the ischemic myocardium damage model of anesthetized rats, as
seen in Table 4, the vehicle-treated group showed a myocardial
infarction rate to area at risk (IZ/AAR) of 60.8%, which indicates
a serious damage in the myocardial muscle. Being measured to be
39.1% in myocardial infarction rate, BMS-180448 showed noticeable
anti-ischemic activity. When compared only in myocardial infarction
rate, the compound of example 1 was similar to BMS-180448. However,
because the compound of example 1 is remarkably lower in
vasodilatation activity (IC.sub.50=9.83 .mu.M) than is BMS-180448
(IC.sub.50=1.38 .mu.M), it is superior to the conventional drug in
cardioselective anti-ischemic activity. Further, the compounds of
the present invention did not act to reduce the blood pressure in
this experiment. Then, the compounds of the present invention can
be used as a curative for the treatment of ischemic heart diseases
by virtue of their excellent protective activity against ischemic
cardiovascular diseases such as such as myocardial infarction,
heart failure, and angina pectoris, etc.
Experimental Example 10
Acute Oral Toxicity Test in Rats
The test to confirm the toxicity of the compounds of formula 1 was
carried out as follows.
In this test six-week old SPF SD rats were used with two rats
assigned to each group. The compounds of examples 1-37 were
suspended in 0.5% methyl cellulose, respectively, and administered
orally in a single dose with 10 ml/kg/15 mL. After the
administration, the animals were observed for clinical signs of
toxicity or mortality and the body weight changes were measured.
All survivors at the end of the observation period underwent
laparotomy under ether anesthesia and the blood samples were taken
from the abdominal aorta for hematological tests and biochemical
analysis. After sacrificing the animals, autopsy was performed for
macroscopic observation of the organs and tissues. Tissue samples
of vital organs from macroscopic legion were removed and fixed in
10% neutral buffered formalin solution, then processed by standard
procedures for histopathology and examined under light microscope.
There were no significant changes in clinical symptoms, body weight
and mortality. Also in hematology, serum chemistry parameters and
macroscopic observation, no drug-related changes were observed. As
a result all the compounds tested did not show toxicity in rats up
to a dose of 10 mg/kg, and the lethal dose (LD.sub.50) for oral
administration was determined to be over 100 mg/kg in rats.
The present invention has been described in an illustrative manner,
and it is to be understood that the terminology used is intended to
be in the nature of description rather than of limitation. Many
modifications and variations of the present invention are possible
in light of the above teachings. Therefore, it is to be understood
that within the scope of the appended claims, the invention may be
practiced otherwise than as specifically described.
Formulation Examples
The pharmaceutical composition containing the compound of formula 1
as an active ingredient can be administered orally or parenterally.
The method for preparation of a tablet for oral administration and
the method for powders and capsules, and the method for an
injection solution for parenteral administration are illustrated as
the followings.
Formulation Example 1
Preparation of a Tablet (Direct Pressure)
The tablet containing 5 mg of the compound of formula 1 as an
active ingredient was prepared as the followings. 5 mg of the
compound was ground and passed through a sieve, and was mixed with
14.1 mg of lactose, 0.8 mg of crossphobidone USNF, and 0.1 mg of
magnesium stearate. The resultant mixture was made into the tablet
under pressure.
Formulation Example 2
Preparation of a Tablet
The tablet containing 5 mg of the compound of formula 1 as an
active ingredient was prepared as the followings.
5 mg of the compound was passed through a sieve, and mixed with
16.0 mg of lactose, 4.0 mg of starch, and then an appropriate
amount of polysorbate 80 (0.3 mg) solution was added. The resultant
mixture was ground and passed through a sieve and then dried.
Colloidal silicon dioxide and 2.0 mg of magnesium stearate were
added and blended. The resultant mixture was made into the tablet
by conventional method.
Formulation Example 3
Preparation of a Powder and Capsule
5 mg of the compound was passed through a sieve, and mixed with
14.8 mg of lactose, 10.0 mg of polyvinyl pyrrolidone, 0.2 mg of
magnesium stearate. The resultant mixture was blended, and filled
into a gelatin capsule (No. 5) using an appropriate instrument by
conventional method.
Formulation Example 4
Preparation of an Injection Solution
The injection solution containing 100 mg of the compound of formula
1 as an active ingredient, and 180 mg of mannitol, 26 mg of
Na.sub.2HPO.sub.4.12H.sub.2O in 2974 mg of distilled water, was
prepared.
* * * * *